The ontogeny of the human globin genes is an important focus of study both with respect to the fundamental developmental biology and molecular genetics of the control of expression of this complex gene system, but also because modifying this developmental control would be of therapeutic value in the treatment of the prevalent genetic diseases of hemoglobin. We are studying this problem from several aspects. First, we have improved culture methods for human erythroid precursors, by antibody selection, so as to obtain purified populations of these cells to establish at which developmental stage drugs change their phenotype. Second, we have developed real-time, quantitative PCR methods to measure globin gene expression in single cells and have used these methods to analyze the mechanisms of the effects of chemically useful drugs, such as hydroxyurea and butyrate, on globin gene expression. Third, we have recently shown that nitric oxide donors, such as cysteine-NO, can induce fetal hemoglobin expression in K562 cells as well as the purified erythroid (CD34) precursor cells described above. Induction of fetal hemoglobin was demonstrated both by quantitative PCR of gamma-globin mRNA and by HPLC of fetal hemoglobin protein. Further, we have shown that both this agent and hydroxyurea acts by stimulating guanylyl cyclase and increasing cyclic GMP levels; conversely, inhibitors of guanylyl cyclase block the action of both agents. These results raise the possiblity that agents which affect this cyclic nucleotide pathway can be used to increase fetal hemoglobin levels in patients with sickle cell anemia and thalassemia. In related studies, we have shown that erythroid cells can be separated in a magnetic field depending on the magnetic susceptibility of the intrinsic hemoglobin. This magnetophoresis technique has been used with mature erythocytes up to now but we are trying to extend it to the study of nucleated erythroid cells.